Treatment of von willebrand disease

ABSTRACT

Provided herein are methods and compositions for treating von Willebrand disease with platelets, platelet derivatives, and/or thrombosomes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/969,942, filed on Feb. 4, 2020, U.S. Provisional PatentApplication No. 62/980,850, filed on Feb. 24, 2020, and U.S. ProvisionalPatent Application No. 63/065,337, filed on Aug. 13, 2020, the contentsof which are incorporated herein by reference in their entireties.

TECHNICAL FIELD

Provided herein, are methods of treating conditions, such von Willebranddisease with platelets, platelet derivatives, and/or thrombosomes. Insome embodiments, the platelets, platelet derivatives, and/orthrombosomes are loaded with anti-fibrinolytic compounds.

Anti-fibrinolytic loaded platelets described herein can be stored undertypical ambient conditions, refrigerated, cryopreserved, for examplewith dimethyl sulfoxide (DMSO), and/or lyophilized after stabilization(e.g., to form thrombosomes)

BACKGROUND

Blood is a complex mixture of numerous components. In general, blood canbe described as comprising four main parts: red blood cells, white bloodcells, platelets, and plasma. The first three are cellular or cell-likecomponents, whereas the fourth (plasma) is a liquid component comprisinga wide and variable mixture of salts, proteins, and other factorsnecessary for numerous bodily functions. The components of blood can beseparated from each other by various methods. In general, differentialcentrifugation is most commonly used currently to separate the differentcomponents of blood based on size and, in some applications, density.

Unactivated platelets, which are also commonly referred to asthrombocytes, are small, often irregularly-shaped (e.g., discoidal orovoidal) megakaryocyte-derived components of blood that are involved inthe clotting process. They aid in protecting the body from excessiveblood loss due not only to trauma or injury, but to normal physiologicalactivity as well.

Platelets are considered crucial in normal hemostasis, providing thefirst line of defense against blood escaping from injured blood vessels.Platelets generally function by adhering to the lining of broken bloodvessels, in the process becoming activated, changing to an amorphousshape, and interacting with components of the clotting system that arepresent in plasma or are released by the platelets themselves or othercomponents of the blood. Purified platelets have found use in treatingsubjects with low platelet count (thrombocytopenia) and abnormalplatelet function (thrombasthenia). Concentrated platelets are oftenused to control bleeding after injury or during acquired plateletfunction defects or deficiencies, for example those occurring duringsurgery and those due to the presence of platelet inhibitors.

SUMMARY OF THE INVENTION

Provided herein are methods and compositions that can be used to treatVon Willebrand disease (VWD) with thrombosomes (e.g., unloadedthrombosomes). Also, provided herein are methods of treating vonWillebrand disease in a subject, including administering atherapeutically effective amount of anti-fibrinolytic loaded plateletsto the subject in need thereof. Also provided herein are methods oftreating von Willebrand disease where the method does not compriseadministering an anti-fibrinolytic (or other therapeutic agent).

Provided herein are methods of treating a hemorrhage in a subject,including administering a therapeutically effective amount ofanti-fibrinolytic loaded platelets to the subject in need thereof.

In some embodiments of any of the methods provided herein, theconcentration of the therapeutically effective amount ofanti-fibrinolytic loaded into the platelets is from about 100 μM toabout 10 mM.

In some embodiments of any of the methods described herein, theanti-fibrinolytic is selected from the group including of ε-aminocaproicacid, aprotinin, aminomethylbenzoic acid, tranexamic acid, andfibrinogen.

In some embodiments, the anti-fibrinolytic is ε-aminocaproic acid. Insome embodiments, the ε-aminocaproic acid is present in a concentrationfrom about 1 μM to about 100 mM.

Also provided herein are methods of treating a hemorrhage in a subject,including administering a therapeutically effective amount of unloadedthrombosomes to the subject in need thereof. In some embodiments oftreating a hemorrhage in a subject, the concentration of thetherapeutically effective amount of unloaded thrombosomes is from about1×10² particles/kg to about 1×10¹³ particles/kg.

In some embodiments of treating a coagulopathy in a subject, thecomposition is administered following administration to the subject anantiplatelet agent or an anticoagulant, or a subject having VonWillebrand disease.

Also provided herein are methods of treating von Willebrand disease in asubject, the method comprising: administering a therapeuticallyeffective amount of freeze-dried platelets to the subject in needthereof.

Also provided herein are methods of treating von Willebrand disease in asubject, the method comprising: administering a therapeuticallyeffective amount of freeze-dried platelets to the subject, wherein themethod does not comprise administering an anti-fibrinolytic.

In some embodiments, the von Willebrand disease is von Willebranddisease type 1, von Willebrand disease type 2, or von Willebrand diseasetype 3. In some embodiments, the von Willebrand disease is acquired vonWillebrand disease.

In some the concentration of the therapeutically effective amount offreeze-dried platelets is from about 1×10² particles/kg to about 1×10¹³particles/kg. In some embodiments, the concentration of thetherapeutically effective amount of freeze-dried platelets is from about1×10⁴ to about 1×10¹¹ particles/kg. In some embodiments, theconcentration of the therapeutically effective amount of freeze-driedplatelets is from about 1×10⁶ to about 1×10⁹ particles/kg. In someembodiments, the concentration of the therapeutically effective amountof freeze-dried platelets is at least 8.5×10⁸ particles/kg. In someembodiments, the concentration of the therapeutically effective amountof freeze-dried platelets is at least 8.49×10⁹ particles/kg.

In some embodiments, the surface expression of CD42b on thetherapeutically effective amount of freeze-dried platelets is about 50%less than the surface expression of CD42b on platelets. In someembodiments, the surface expression of CD42b on the therapeuticallyeffective amount of freeze-dried platelets is about 40% less than thesurface expression of CD42b on platelets. In some embodiments, thesurface expression of CD42b on the therapeutically effective amount offreeze-dried platelets is about 25% less than the surface expression ofCD42b on platelets. In some embodiments, the therapeutically effectiveamount of freeze-dried platelets forms clots in von Willebrand factordeficient plasma.

In some embodiments, the therapeutically effective amount offreeze-dried platelets are administered topically. In some embodiments,the therapeutically effective amount of freeze-dried platelets areadministered intravenously. In some embodiments, therapeuticallyeffective amount of freeze-dried platelets are administeredintramuscularly. In some embodiments, the therapeutically effectiveamount of freeze-dried platelets are administered subcutaneously.

Also provided herein are methods of treating a coagulopathy in asubject, the method comprising administering to the subject in needthereof an effective amount of a composition comprising platelets orplatelet derivatives and an incubating agent comprising one or moresalts, a buffer, optionally a cryoprotectant, and optionally an organicsolvent, wherein the composition is administered to the subject havingvon Willebrand disease.

Also provided herein are methods of treating a coagulopathy in asubject, the method comprising administering to the subject in needthereof a therapeutically effective amount of a composition prepared bya process comprising incubating platelets with an incubating agentcomprising one or more salts, a buffer, optionally a cryoprotectant, andoptionally an organic solvent, to form the composition, wherein thecomposition is administered to the subject having von Willebranddisease.

In some embodiments, the von Willebrand disease is von Willebranddisease type 1, von Willebrand disease type 2, von Willebrand diseasetype 3, or acquired von Willebrand disease.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing aggregation slope of six separate lots ofthrombosomes (A-F) compared to formalin-fixed platelets (positivecontrol) in a ristocetin aggregation assay. Ristocetin failed toaggregate thrombosomes and all thrombosomes lots (A-F) weresignificantly different from the fixed platelet positive control.

FIG. 2 is a graph showing aggregation of thrombosomes in plasma ascompared to platelet rich plasma.

FIG. 3 is a graph showing CD42b expression in platelets as compared tothrombosomes.

FIG. 4 is a graph showing T-TAS occlusion data under shear stress ofplatelets and thrombosomes in plasma.

FIG. 5 is a graph showing T-TAS occlusion data under shear stress ofthrombosomes in normal plasma and von Willebrand factor (vWF) deficientplasma.

DETAILED DESCRIPTION

This disclosure is directed to compositions and methods for use ofplatelets, platelet derivatives, or thrombosomes as biological carriersof cargo, such as anti-fibrinolytic compounds, also referred to hereinas anti-fibrinolytic loaded platelets, platelet derivatives, orthrombosomes. This disclosure is also directed to compositions andmethods for use of unloaded platelets, platelet derivatives, orthrombosomes in the treatment of a disease such as von Willebranddisease, or conditions such as hemorrhaging or trauma.

Anti-fibrinolytic loaded platelets described herein can be stored undertypical ambient conditions, refrigerated, cryopreserved, for examplewith dimethyl sulfoxide (DMSO), and/or lyophilized after stabilization(e.g., to form thrombosomes).

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. Further, where a range of values is disclosed, theskilled artisan will understand that all other specific values withinthe disclosed range are inherently disclosed by these values and theranges they represent without the need to disclose each specific valueor range herein. For example, a disclosed range of 1-10 includes 1-9,1-5, 2-10, 3.1-6, 1, 2, 3, 4, 5, and so forth. In addition, eachdisclosed range includes up to 5% lower for the lower value of the rangeand up to 5% higher for the higher value of the range. For example, adisclosed range of 4-10 includes 3.8-10.5. This concept is captured inthis document by the term “about”.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a platelet” includes aplurality of such platelets. Furthermore, the use of terms that can bedescribed using equivalent terms include the use of those equivalentterms. Thus, for example, the use of the term “subject” is to beunderstood to include the terms “patient”, “individual,” or “animal” andother terms used in the art to indicate one who is subject to atreatment.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” contemplate an action that occurs while asubject is suffering from a disease (e.g., von Willebrand disease),disorder, and/or condition (e.g., hemorrhage) which reduces the severityof the disease, disorder, and/or conditions or slows the progression ofthe disease, disorder, or condition (“therapeutic treatment”), and whichcan inhibit the disease, disorder, and/or condition (e.g., hemorrhage).

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of is an amount sufficient to provide a therapeuticbenefit in the treatment of the disease, disorder and/or condition(e.g., hemorrhage) or to delay or minimize one or more symptomsassociated with the disease, disorder, and/or condition. Atherapeutically effective amount means an amount of therapeutic agent,alone or in combination with other therapies, which provides atherapeutic benefit in the treatment of the disease, disorder, and/orcondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms orcauses of the disease, disorder and/or condition, or enhances thetherapeutic efficacy of another therapeutic agent.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the term belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present disclosure, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.The present disclosure is controlling to the extent it conflicts withany incorporated publication.

As used herein and in the appended claims, the term “platelet” caninclude whole platelets, fragmented platelets, platelet derivatives, orthrombosomes. “Platelets” within the above definition may include, forexample, platelets in whole blood, platelets in plasma, platelets inbuffer optionally supplemented with select plasma proteins, cold storedplatelets, dried platelets, cryopreserved platelets, thawedcryopreserved platelets, rehydrated dried platelets, rehydratedcryopreserved platelets, lyopreserved platelets, thawed lyopreservedplatelets, or rehydrated lyopreserved platelets. “Platelets” may be“platelets” of mammals, such as of humans, or such as of non-humanmammals.

Thus, for example, reference to “anti-fibrinolytic loaded platelets” maybe inclusive of anti-fibrinolytic loaded platelets as well asanti-fibrinolytic loaded platelet derivatives or anti-fibrinolyticloaded thrombosomes, unless the context clearly dictates a particularform.

As used herein, “thrombosomes” (sometimes also herein called “Tsomes” or“Ts”, particularly in the Examples and Figures) are platelet derivativesthat have been treated with an incubating agent (e.g., any of theincubating agents described herein) and lyopreserved (e.g., freeze-driedto form thrombosomes). In some cases, thrombosomes can be prepared frompooled platelets. Thrombosomes can have a shelf life of 2-3 years in dryform at ambient temperature and can be rehydrated with sterile waterwithin minutes for immediate infusion.

As used herein and in the appended claims, the term “fresh platelet”includes platelets stored for less than approximately 24 hours.

As used herein and in the appended claims the term “stored platelet”includes platelets stored for approximately 24 hours or longer beforeuse.

As used herein and in the appended claims the term “fixed platelet”includes platelets fixed with a formalin solution.

As used herein and in the appended claims the term “unloaded” includesplatelets, platelet derivatives, and/or thrombosomes that are not loadedwith an active agent, such as platelets, platelet derivatives, and/orthrombosomes that are not loaded with an anti-fibrinolytic.

In some embodiments, rehydrating the anti-fibrinolytic loaded plateletsincludes adding to the platelets an aqueous liquid. In some embodiments,the aqueous liquid is water. In some embodiments, the aqueous liquid isan aqueous solution. In some embodiments, the aqueous liquid is a salinesolution. In some embodiments, the aqueous liquid is a suspension.

In some embodiments, the rehydrated platelets have coagulation factorlevels showing all individual factors (e.g., Factors VII, VIII and IX)associated with blood clotting at 40 international units (IU) orgreater.

As used herein, “coagulopathy” is a bleeding disorder in which theblood's ability to coagulate (e.g., form clots) is impaired. Thiscondition can cause a tendency toward prolonged or excessive bleed(e.g., diathesis). In some embodiments, a coagulopathy is caused by adisease (e.g., Von Willebrand disease). In some embodiments, acoagulopathy is a drug induced coagulopathy. In some embodiments, acoagulopathy is induced by an antiplatelet agent-induced coagulopathy.In some embodiments, a coagulopathy is induced by an anti-plateletagent.

In some embodiments, the dried platelets, such as freeze-driedplatelets, have less than about 10%, such as less than about 8%, such asless than about 6%, such as less than about 4%, such as less than about2%, such as less than about 0.5% crosslinking of platelet membranes viaproteins and/or lipids present on the membranes. In some embodiments,the dried platelets, such as freeze dried platelets, have less thanabout 10%, such as less than about 8%, such as less than about 6%, suchas less than about 4%, such as less than about 2%, such as less thanabout 0.5% crosslinking of platelet membranes via proteins and/or lipidspresent on the membranes. In some embodiments, the rehydrated platelets,have less than about 10%, such as less than about 8%, such as less thanabout 6%, such as less than about 4%, such as less than about 2%, suchas less than about 0.5% crosslinking of platelet membranes via proteinsand/or lipids present on the membranes. In some embodiments, therehydrated platelets, have between about 0.01% to about 5%, such asbetween about 0.1% to about 4%, such as between about 1% to betweenabout 3%, such as between about 1% to about 2%, crosslinking of plateletmembranes via proteins and/or lipids present on the membranes. In someembodiments, the rehydrated platelets, have at least about 1% to atleast about 10, such as less than about 8%, such as less than about 6%,such as less than about 4%, such as less than about 2%, such as lessthan about 0.5% crosslinking of platelet membranes via proteins and/orlipids present on the membranes.

In some embodiments, the anti-fibrinolytic loaded platelets and thedried platelets, such as freeze-dried platelets, having a particle size(e.g., diameter, max dimension) of at least about 0.2 μm (e.g., at leastabout 0.3 μm, at least about 0.4 μm, at least about 0.5 μm, at leastabout 0.6 μm, at least about 0.7 μm, at least about 0.8 μm, at leastabout 0.9 μm, at least about 1.0 μm, at least about 1.0 μm, at leastabout 1.5 μm, at least about 2.0 μm, at least about 2.5 μm, or at leastabout 5.0 μm). In some embodiments, the particle size is less than about5.0 μm (e.g., less than about 2.5 μm, less than about 2.0 μm, less thanabout 1.5 μm, less than about 1.0 μm, less than about 0.9 μm, less thanabout 0.8 μm, less than about 0.7 μm, less than about 0.6 μm, less thanabout 0.5 μm, less than about 0.4 μm, or less than about 0.3 μm). Insome embodiments, the particle size is from about 0.3 μm to about 5.0 μm(e.g., from about 0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8μm).

In some embodiments, at least 50% (e.g., at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or at least about 99%) of platelets and/or the driedplatelets, such as freeze-dried platelets, have a particle size in therange of about 0.3 μm to about 5.0 μm (e.g., from about 0.4 μm to about4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm to about2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm to about0.9 μm, or from about 0.6 μm to about 0.8 μm). In some embodiments, atmost 99% (e.g., at most about 95%, at most about 80%, at most about 75%,at most about 70%, at most about 65%, at most about 60%, at most about55%, or at most about 50%) of platelets and/or the dried platelets, suchas freeze-dried platelets, are in the range of about 0.3 μm to about 5.0μm (e.g., from about 0.4 μm to about 4.0 μm, from about 0.5 μm to about2.5 μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about1.0 μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about0.8 μm). In some embodiments, about 50% to about 99% (e.g., about 55% toabout 95%, about 60% to about 90%, about 65% to about 85, about 70% toabout 80%) of platelets and/or the dried platelets, such as freeze-driedplatelets, are in the range of about 0.3 μm to about 5.0 μm (e.g., fromabout 0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, fromabout 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0 μm, fromabout 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8 μm).

In some embodiments, (e.g., using unloaded platelets or plateletderivatives), the platelets or platelet derivatives are preparedconsistent with the procedures described in U.S. Pat. No. 8,486,617(such as, e.g., Examples 1-5) and U.S. Pat. No. 8,097,403 (such as,e.g., Examples 1-3).

Also provided herein are methods of preparing anti-fibrinolytic loadedplatelets. In some embodiments, platelets are isolated prior tocontacting the platelets with an anti-fibrinolytic.

Accordingly, in some embodiments, the methods for preparinganti-fibrinolytic loaded platelets includes: step (a) isolatingplatelets, for example in a liquid medium; and step (b) contacting theplatelets with an anti-fibrinolytic, and with a loading buffercomprising a salt, a base, a loading agent, and optionally ethanol, toform the anti-fibrinolytic loaded platelets.

Accordingly, in some embodiments, the methods for preparinganti-fibrinolytic loaded platelets includes: step (a) isolatingplatelets, for example in a liquid medium; step (b) contacting theplatelets with an anti-fibrinolytic to form a first composition; andstep (c) contacting the first composition with a buffer comprising asalt, a base, a loading agent, and optionally at least one organicsolvent to form the anti-fibrinolytic loaded platelets.

In some embodiments, suitable organic solvents include, but are notlimited to alcohols, esters, ketones, ethers, halogenated solvents,hydrocarbons, nitriles, glycols, alkyl nitrates, water or mixturesthereof. In some embodiments, suitable organic solvents includes, butare not limited to methanol, ethanol, n-propanol, isopropanol, aceticacid, acetone, methyl ethyl ketone, methyl isobutyl ketone, methylacetate, ethyl acetate, isopropyl acetate, tetrahydrofuran, isopropylether (IPE), tert-butyl methyl ether, dioxane (e.g., 1,4-dioxane),acetonitrile, propionitrile, methylene chloride, chloroform, toluene,anisole, cyclohexane, hexane, heptane, ethylene glycol, nitromethane,dimethylformamide, dimethyl sulfoxide, N-methyl pyrrolidone,dimethylacetamide, and combinations thereof.

Accordingly, in some embodiments, the methods for preparinganti-fibrinolytic loaded platelets includes: step (a) isolatingplatelets, for example in a liquid medium; step (b) contacting theplatelets with a buffer comprising a salt, a base, a loading agent, andoptionally at least one organic solvent, to form a first composition;and step (c) contacting the first composition with an anti-fibrinolytic,to form the anti-fibrinolytic loaded platelets.

In some embodiments, isolating platelets includes isolating plateletsfrom blood.

In some embodiments, platelets are donor-derived platelets. In someembodiments, platelets are obtained by a process that includes anapheresis step. In some embodiments, platelets are fresh platelets. Insome embodiments, platelets are stored platelets.

In some embodiments, platelets are derived in vitro. In someembodiments, platelets are derived or prepared in a culture prior tocontacting the platelets with an anti-fibrinolytic. In some embodiments,preparing the platelets includes deriving or growing the platelets froma culture of megakaryocytes. In some embodiments, preparing theplatelets includes deriving or growing the platelets (or megakaryocytes)from a culture of human pluripotent stem cells (PCSs), includingembryonic stem cells (ESCs) and/or induced pluripotent stem cells(iPSCs).

In some embodiments, the loading agent is a saccharide. In someembodiments, the saccharide is a monosaccharide. In some embodiments,the saccharide is a disaccharide. In some embodiments, the saccharide isa non-reducing disaccharide. In some embodiments, the saccharide issucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, orxylose. In some embodiments, the loading agent is a starch. In someembodiments, a loading agent is a cryoprotectant. In some embodiments,(e.g., for platelets or platelet derivatives not loaded with ananti-fibrinolytic agent), a “loading agent” can be used in thepreparation of the platelets or platelet derivatives, for example, aspart of an incubating agent.

As used herein, the term “anti-fibrinolytic,” “anti-fibrinolytics,” or“anti-fibrinolytic compound,” is any compound capable of inhibitingfibrinolysis. Fibrinolysis is the process where the activatedplasminogen removes excess fibrin and promotes fibrin clot formation andwound healing (Szekely, A. and Lex, D. J., Antifibrinolytics, Heart LungVessel, 6(1): 5-7, (2014), which is incorporated herein by reference inits entirety). Inhibiting fibrinolysis can be useful under certainconditions. For example, in the case of traumatic bleeding events and/orhemorrhage, inhibiting fibrinolysis can enhance the formation of bloodclots (e.g., stopping bleeding).

In some embodiments, the anti-fibrinolytic can be ε-aminocaproic acid.In some embodiments, the anti-fibrinolytic can be tranexamic acid. Insome embodiments, the anti-fibrinolytic can be aprotinin. In someembodiments, the anti-fibrinolytic can be aminomethylbenzoic acid. Insome embodiments, the anti-fibrinolytic can be fibrinogen. In someembodiments, the anti-fibrinolytic can be a combination of two or moreanti-fibrinolytics.

Provided herein are methods to treat acquired von Willebrand disease(e.g., any of the von Willebrand diseases described herein), comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising contactingthrombosomes with a loading buffer including a salt, a base, a loadingagent, and optionally at least one organic solvent, and a step offreeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes prepared bya process comprising providing platelets and contacting the plateletswith a loading buffer including a salt, a base, a loading agent, andoptionally at least one organic solvent, and a step of freeze-drying, toform the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising contactingplatelets with a loading buffer including a salt and a base to form afirst composition and contacting the first composition with a loadingagent, and optionally at least one organic solvent, and a step offreeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising contactingplatelets with a loading agent, and optionally at least one organicsolvent to form a first composition and contacting the first compositionwith a loading buffer including a salt and a base, and a freeze-dryingstep, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising contactingplatelets a loading buffer including a salt, a base, a loading agent,and optionally at least one organic solvent, and a step of freeze-dryingto form the anti-fibrinolytic-loaded thrombosomes. In some embodimentsof preparing unloaded thrombosomes, the platelets are pooled from aplurality of donors prior to a treating step.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising A) poolingplatelets from a plurality of donors and B) contacting the plateletsfrom step (A) with a loading buffer including a salt, a base, a loadingagent, and optionally at least one organic solvent, and a freeze-dryingstep, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising A) poolingplatelets from a plurality of donors and B) contacting the plateletsfrom step (A) with a loading buffer including a salt and a base to forma first composition and contacting the first composition with a loadingagent, and optionally at least one organic solvent, and a step offreeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising A) poolingplatelets from a plurality of donors and B) contacting the plateletsfrom step (A) with an a loading agent to form a first composition andcontacting the first composition with a loading buffer including a saltand a base, and optionally at least one organic solvent, and a step offreeze-drying, to form the unloaded thrombosomes.

In some embodiments, no solvent is used. Thus, provided herein aremethods to treat von Willebrand disease, comprising a therapeuticallyeffective amount of unloaded thrombosomes, wherein the unloadedthrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;    -   B) contacting the platelets with an unloaded and with a loading        buffer comprising a salt, a base, and a loading agent, to form        the unloaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol, and    -   C) a step of freeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;    -   B) contacting the platelets with an unloaded to form a first        composition;    -   C) contacting the first composition with a buffer comprising a        salt, a base, and a loading agent, to form the unloaded        platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol, and    -   (D) a step freeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;    -   B) contacting the platelets with a buffer comprising a salt and        a base, to form a first composition;    -   C) contacting the first composition with a loading agent, to        form the unloaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol and    -   D) a step of freeze drying, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising:

-   -   A) preparing platelets;    -   B) contacting the platelets with an anti-fibrinolytic and with a        loading buffer comprising a salt, a base, and a loading agent,        to form the unloaded platelets,        -   wherein the method does not comprise contacting the            platelets with an organic solvent such as ethanol, and    -   C) a step of freeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of unloaded thrombosomes, wherein theunloaded thrombosomes are prepared by a process comprising:

a) preparing platelets;

b) contacting the platelets with a loading agent to form a firstcomposition;

c) contacting the first composition with a buffer comprising a salt anda base, to form the unloaded platelets,

-   -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol and

d) a step of freeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic loaded thrombosomes areprepared by a process comprising contacting thrombosomes with ananti-fibrinolytic and with a loading buffer including a salt, a base, aloading agent, and optionally at least one organic solvent, and a stepof freeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes prepared by a process comprising providing platelets andcontacting the platelets with an anti-fibrinolytic and with a loadingbuffer including a salt, a base, a loading agent, and optionally atleast one organic solvent, and a step of freeze-drying, to form theanti-fibrinolytic loaded thrombosomes. In some embodiments of preparinganti-fibrinolytic loaded platelets, the platelets are contacted with theanti-fibrinolytic and with the loading buffer sequentially, in eitherorder.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic loaded thrombosomes areprepared by a process comprising contacting platelets with theanti-fibrinolytic to form a first composition and contacting the firstcomposition with a loading buffer including a salt, a base, a loadingagent, and optionally at least one organic solvent, and a step offreeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic loaded thrombosomes areprepared by a process comprising contacting platelets with a bufferincluding a salt, a base, a loading agent, and optionally at least oneorganic solvent to form a first composition and contacting the firstcomposition with an anti-fibrinolytic, and a freeze drying step, to formthe anti-fibrinolytic loaded thrombosomes. In some embodiments ofpreparing anti-fibrinolytic loaded thrombosomes, the platelets arecontacted with the anti-fibrinolytic and with the loading bufferconcurrently.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic loaded thrombosomes areprepared by a process comprising contacting platelets with ananti-fibrinolytic in the presence of a loading buffer including a salt,a base, a loading agent, and optionally at least one organic solvent,and a step of freeze-drying to form the anti-fibrinolytic-loadedthrombosomes. In some embodiments of preparing anti-fibrinolytic loadedthrombosomes, the platelets are pooled from a plurality of donors priorto a treating step.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic loaded thrombosomes areprepared by a process comprising A) pooling platelets from a pluralityof donors and B) contacting the platelets from step (A) with ananti-fibrinolytic and with a loading buffer including a salt, a base, aloading agent, and optionally at least one organic solvent, and afreeze-drying step, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic loaded thrombosomes areprepared by a process comprising A) pooling platelets from a pluralityof donors and B) contacting the platelets from step (A) with ananti-fibrinolytic to form a first composition and contacting the firstcomposition with a loading buffer including a salt, a base, a loadingagent, and optionally at least one organic solvent, and a step offreeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic loaded thrombosomes areprepared by a process comprising A) pooling platelets from a pluralityof donors and B) contacting the platelets from step (A) with ananti-fibrinolytic to form a first composition and contacting the firstcomposition with a loading buffer including a salt, a base, a loadingagent, and optionally at least one organic solvent, and a step offreeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic loaded thrombosomes areprepared by a process comprising A) pooling platelets from a pluralityof donors and B) contacting the platelets from step (A) with a loadingbuffer including a salt, a base, a loading agent, and optionally atleast one organic solvent, to form a first composition and contactingthe first composition with an anti-fibrinolytic, and a step offreeze-drying to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat von Willebrand disease (e.g., anyof the von Willebrand diseases described herein), comprising atherapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic loaded thrombosomes areprepared by a process comprising A) pooling platelets from a pluralityof donors and B) contacting the platelets with an anti-fibrinolytic inthe presence of a loading buffer including a salt, a base, a loadingagent, and optionally at least one organic solvent, and a step offreeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

In some embodiments, no solvent is used. Thus, provided herein aremethods to treat von Willebrand disease, comprising a therapeuticallyeffective amount of anti-fibrinolytic loaded thrombosomes, wherein theanti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;    -   D) contacting the platelets with an anti-fibrinolytic and with a        loading buffer comprising a salt, a base, and a loading agent,        to form the anti-fibrinolytic loaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol, and    -   C) a step of freeze-drying, to form the anti-fibrinolytic loaded        thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared bya process comprising:

-   -   A) isolating platelets, for example in a liquid medium;    -   B) contacting the platelets with an anti-fibrinolytic to form a        first composition;    -   E) contacting the first composition with a buffer comprising a        salt, a base, and a loading agent, to form the anti-fibrinolytic        loaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol, and    -   (D) a step freeze-drying, to form the anti-fibrinolytic loaded        thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared bya process comprising:

-   -   A) isolating platelets, for example in a liquid medium;    -   B) contacting the platelets with a buffer comprising a salt, a        base, and a loading agent, to form a first composition;    -   C) contacting the first composition with an anti-fibrinolytic,        to form the anti-fibrinolytic loaded platelets,    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol and    -   D) a step of freeze drying, to form the anti-fibrinolytic loaded        thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared bya process comprising:

-   -   A) preparing platelets;    -   D) contacting the platelets with an anti-fibrinolytic and with a        loading buffer comprising a salt, a base, and a loading agent,        to form the anti-fibrinolytic loaded platelets,        -   wherein the method does not comprise contacting the            platelets with an organic solvent such as ethanol, and    -   E) a step of freeze-drying, to form the anti-fibrinolytic loaded        thrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared bya process comprising:

a) preparing platelets;

b) contacting the platelets with an anti-fibrinolytic to form a firstcomposition;

c) contacting the first composition with a buffer comprising a salt, abase, and a loading agent, to form the anti-fibrinolytic loadedplatelets,

-   -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol and

d) a step of freeze-drying, to form the anti-fibrinolytic loadedthrombosomes.

Provided herein are methods to treat von Willebrand disease, comprisinga therapeutically effective amount of anti-fibrinolytic loadedthrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared bya process comprising:

-   -   a) preparing platelets;    -   b) contacting the platelets with a buffer comprising a salt, a        base, and a loading agent, to form a first composition;    -   c) contacting the first composition with an anti-fibrinolytic,        to form the anti-fibrinolytic loaded platelets.    -   wherein the method does not comprise contacting the platelets        with an organic solvent such as ethanol and the method does not        comprise contacting the first composition with an organic        solvent such as ethanol and    -   d) a freeze-drying step, to form the anti-fibrinolytic loaded        thrombosomes.

In some embodiments, an anti-fibrinolytic (e.g., EACA) loaded intoplatelets is modified to include an imaging agent. For example, ananti-fibrinolytic can be modified with an imaging agent in order toimage the anti-fibrinolytic loaded platelet in vivo. In someembodiments, an anti-fibrinolytic can be modified with two or moreimaging agents (e.g., any two or more of the imaging agents describedherein). In some embodiments, an anti-fibrinolytic loaded into plateletsis modified with a radioactive metal ion, a paramagnetic metal ion, agamma-emitting radioactive halogen, a positron-emitting radioactivenon-metal, a hyperpolarized NMR-active nucleus, a reporter suitable forin vivo optical imaging, or a beta-emitter suitable for intravasculardetection. For example, a radioactive metal ion can include, but is notlimited to, positron emitters such as ⁵⁴Cu, ⁴⁸V, ⁵²Fe, ⁵⁵Co, ⁹⁴Tc or⁶⁸Ga; or gamma-emitters such as ¹⁷¹Tc, ¹¹¹In, ¹¹³In, or ⁶⁷Ga. Forexample, a paramagnetic metal ion can include, but is not limited toGd(III), a Mn(II), a Cu(II), a Cr(III), a Fe(III), a Co(II), a Er(II), aNi(II), a Eu(III) or a Dy(III), an element comprising an Fe element, aneodymium iron oxide (NdFeO3) or a dysprosium iron oxide (DyFeO3). Forexample, a paramagnetic metal ion can be chelated to a polypeptide or amonocrystalline nanoparticle. For example, a gamma-emitting radioactivehalogen can include, but is not limited to ¹²³I, ¹³¹I or ⁷⁷Br. Forexample, a positron-emitting radioactive non-metal can include, but isnot limited to ¹¹C, ¹³N, ¹⁵O, ¹⁷F, ¹⁸F, ⁷⁵Br, ⁷⁶Br or ¹²⁴I. For example,a hyperpolarized NMR-active nucleus can include, but is not limited to¹³C, ¹⁵N, ¹⁹F, ²⁹Si and ³¹P. For example, a reporter suitable for invivo optical imaging can include, but is not limited to any moietycapable of detection either directly or indirectly in an optical imagingprocedure. For example, the reporter suitable for in vivo opticalimaging can be a light scatterer (e.g., a colored or uncoloredparticle), a light absorber or a light emitter. For example, thereporter can be any reporter that interacts with light in theelectromagnetic spectrum with wavelengths from the ultraviolet to thenear infrared. For example, organic chromophoric and fluorophoricreporters include groups having an extensive delocalized electronsystem, e.g. cyanines, merocyanines, indocyanines, phthalocyanines,naphthalocyanines, triphenylmethines, porphyrins, pyrilium dyes,thiapyrilium dyes, squarylium dyes, croconium dyes, azulenium dyes,indoanilines, benzophenoxazinium dyes, benzothiaphenothiazinium dyes,anthraquinones, napthoquinones, indathrenes, phthaloylacridones, trisphenoquinones, azo dyes, intramolecular and intermolecularcharge-transfer dyes and dye complexes, tropones, tetrazines,b/s(dithiolene) complexes, bιs(benzene-dithiolate) complexes,iodoaniline dyes, b/stS.O-dithiolene) complexes. For example, thereporter can be, but is not limited to a fluorescent, a bioluminescent,or chemiluminescent polypeptide. For example, a fluorescent orchemiluminescent polypeptide is a green florescent protein (GFP), amodified GFP to have different absorption/emission properties, aluciferase, an aequorin, an obelin, a mnemiopsin, a berovin, or aphenanthridinium ester. For example, a reporter can be, but is notlimited to rare earth metals (e.g., europium, samarium, terbium, ordysprosium), or fluorescent nanocrystals (e.g., quantum dots). Forexample, a reporter may be a chromophore that can include, but is notlimited to fluorescein, sulforhodamine 101 (Texas Red), rhodamine B,rhodamine 6G, rhodamine 19, indocyanine green, Cy2, Cy3, Cy3.5, Cy5,Cy5.5, Cy7, Marina Blue, Pacific Blue, Oregon Green 88, Oregon Green514, tetramethylrhodamine, and Alexa Fluor 350, Alexa Fluor 430, AlexaFluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, AlexaFluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680, Alexa Fluor 700, and Alexa Fluor 750. For example, abeta-emitter can include, but is not limited to radio metals ⁶⁷Cu, ⁸⁹Sr,⁹⁰Y, ¹⁵³Sm, ¹⁸⁵Re, ¹⁸⁸Re or ¹⁹²Ir, and non-metals ³²P, ³³P, ³⁸S, ³⁸Cl,³⁹Cl, ⁸²Br and ⁸³Br. In some embodiments, an anti-fibrinolytic loadedinto platelets can be associated with gold or other equivalent metalparticles (such as nanoparticles). For example, a metal particle systemcan include, but is not limited to gold nanoparticles (e.g., Nanogold™).

In some embodiments, an anti-fibrinolytic loaded into platelets that ismodified with an imaging agent is imaged using an imaging unit. Theimaging unit can be configured to image the anti-fibrinolytic loadedplatelets in vivo based on an expected property (e.g., optical propertyfrom the imaging agent) to be characterized. For example, imagingtechniques (in vivo imaging using an imaging unit) that can be used, butare not limited to are: computer assisted tomography (CAT), magneticresonance spectroscopy (MRS), magnetic resonance imaging (MRI), positronemission tomography (PET), single-photon emission computed tomography(SPECT), or bioluminescence imaging (BLI). Chen, Z., et al., Advance ofMolecular Imaging Technology and Targeted Imaging Agent in Imaging andTherapy, Biomed Res Int., 819324, doi: 10.1155/2014/819324 (2014) havedescribed various imaging techniques and which is incorporated byreference herein in its entirety.

In some embodiments, the platelets are isolated prior to a contactingstep. In some embodiments, the methods further include isolatingplatelets by using centrifugation. In some embodiments, thecentrifugation occurs at a relative centrifugal force (RCF) of about 800g to about 2000 g. In some embodiments, the centrifugation occurs atrelative centrifugal force (RCF) of about 1300 g to about 1800 g. Insome embodiments, the centrifugation occurs at relative centrifugalforce (RCF) of about 1500 g. In some embodiments, the centrifugationoccurs for about 1 minute to about 60 minutes. In some embodiments, thecentrifugation occurs for about 10 minutes to about 30 minutes. In someembodiments, the centrifugation occurs for about 20 minutes.

In some embodiments, the platelets are at a concentration from about1,000 platelets/μl to about 10,000,000 platelets/μl. In someembodiments, the platelets are at a concentration from about 50,000platelets/μl to about 4,000,000 platelets/μl. In some embodiments, theplatelets are at a concentration from about 100,000 platelets/μl toabout 300,000,000 platelets/μl. In some embodiments, the platelets areat a concentration from about 1,000,000 to about 2,000,000. In someembodiments, the platelets are at a concentration of about 2,000,000platelets/μl.

In some embodiments, the platelets are at a concentration from about1,000 platelets to about 10,000,000 platelets. In some embodiments, theplatelets are at a concentration from about 50,000 platelets to about4,000,000 platelets. In some embodiments, the platelets are at aconcentration from about 100,000 platelets to about 300,000,000platelets. In some embodiments, the platelets are at a concentrationfrom about 1,000,000 to about 2,000,000. In some embodiments, theplatelets are at a concentration of about 2,000,000 platelets.

Unloaded platelets can be used, for example, in therapeutic applicationsas disclosed herein. For example, unloaded platelets, unloaded plateletderivatives, and/or unloaded thrombosomes can be used to treat adisease, such as von Willebrand disease. In some embodiments, unloadedplatelets, unloaded platelet derivatives, and/or unloaded thrombosomescan be used to treat von Willebrand disease. In some embodiments,unloaded platelets, unloaded platelet derivatives, and/or unloadedthrombosomes can be used to treat in a non-limiting way von Willebranddisease type 1, von Willebrand disease type 2, or von Willebrand diseasetype 3. In some embodiments, unloaded platelets, unloaded plateletderivatives, and/or unloaded thrombosomes can be used to treat acquiredvon Willebrand disease. Generally, acquired von Willebrand diseaseoccurs with an autoimmune disorder (e.g., lupus) or after taking certainmedications.

Von Willebrand disease is a congenital coagulation disorder caused bythe lack or a defect in the gene required to produce active vonWillebrand protein (e.g., von Willebrand factor (vWF). The diseaseaffects about 1% of the population. Von Willebrand disease manifestsitself with patients experiencing frequent nosebleeds, easy bruising,excessive bleeding during menstruation, and/or invasive procedures. vWFis produced in endothelial cells and megakaryocytes and is released intocirculation bound to Factor VIII. Von Willebrand factor assists duringplatelet plug formation by binding both clotting factor VIII andplatelets. The plasma levels of vWF in a human subject are about 1 ug/mLwith a half-life of about two hours. vWF under shear vascular stressbinds to exposed collagen of damaged vascular subendothelium. vWFbinding to the subendothelium collagen bridges platelets binding to thesite of injury. The vWF binds platelets through the GPIbα receptor(CD42b) and the GPIIb-IIIα receptor (CD41/CD61) complex. A subjectlacking functioning vWF protein lack and/or have a reduced ability toclot blood easily, and therefore bleed readily upon injury. In someembodiments, thrombosomes can function as a stabilized platelet productand can participate in clot formation. In some embodiments, thrombosomescan participate in clot formation in the absence of vWF. In someembodiments, bound thrombosomes can help potentiate thrombin productionand strengthen a blood clot.

In some embodiments, the surface expression of CD42b on thetherapeutically effective amount of unloaded thrombosomes is about 50%less than the surface expression of CD42b on platelets. In someembodiments, the surface expression of CD42b on the therapeuticallyeffective amount of unloaded thrombosomes is about 40% less than thesurface expression of CD42b on platelets. In some embodiments, thesurface expression of CD42b on the therapeutically effective amount ofunloaded thrombosomes is about 25% less than the surface expression ofCD42b on platelets. In some embodiments, the surface expression of CD42bon the therapeutically effect amount of unloaded thrombosomes is about50%, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%,about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%,about 30%, about 29%, about 28%, about 27%, about 26%, or about 25%.

In some embodiments, the therapeutically effective amount of unloadedthrombosomes forms clots at about 60% of the time platelets form clots.In some embodiments, the therapeutically effective amount of unloadedthrombosomes forms clots at about 70% of the time platelets form clots.In some embodiments, the therapeutically effective amount of unloadedthrombosomes forms clots at about 80% of the time platelets form. Insome embodiments, the therapeutically effective amount of unloadedthrombosomes forms clots at about 50%, about 51%, about 52%, about 53%,about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%,about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%,or about 80%.

In some embodiments, the therapeutically effective amount of unloadedthrombosomes forms clots at about 60% of the time platelets form clotsin von Willebrand factor deficient plasma. In some embodiments, thetherapeutically effective amount of unloaded thrombosomes forms clots atabout 70% of the time platelets form clots in von Willebrand factordeficient plasma. In some embodiments, the therapeutically effectiveamount of unloaded thrombosomes forms clots at about 80% of the timeplatelets form in von Willebrand factor deficient plasma. In someembodiments, the therapeutically effective amount of unloadedthrombosomes forms clots at about 50%, about 51%, about 52%, about 53%,about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%,about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%,or about 80% in von Willebrand factor deficient plasma. In someembodiments, the therapeutically effective amount of unloadedthrombosomes (e.g., freeze-dried platelets) forms clots in vonWillebrand deficient plasma.

In some embodiments, treatment of a subject with platelets loaded withan anti-fibrinolytic compound provides a “r” time (time to clot) that isshorter than the “r” time for treatment of the subject with the sameamount of the free anti-fibrinolytic compound, that is, theanti-fibrinolytic compound that is not loaded into the platelets.

In some embodiments, treatment of a subject with thrombosomes loadedwith an anti-fibrinolytic compound provides a “r” time (time to clot)that is shorter than the “r” time for treatment of the subject with thesame amount of the free anti-fibrinolytic compound, that is, theanti-fibrinolytic compound that is not loaded into the thrombosomes.

The anti-fibrinolytic loaded platelets can be used in therapeuticapplications as disclosed herein. For example, the anti-fibrinolyticloaded platelets can be used to treat von Willebrand disease (describedherein).

Any known technique for drying platelets can be used in accordance withthe present disclosure, as long as the technique can achieve a finalresidual moisture content of less than 5%. Preferably, the techniqueachieves a final residual moisture content of less than 2%, such as 1%,0.5%, or 0.1%. Non-limiting examples of suitable techniques arefreeze-drying (lyophilization) and spray-drying. A suitablelyophilization method is presented in Table A. Additional exemplarylyophilization methods can be found in U.S. Pat. Nos. 7,811,558,8,486,617, and 8,097,403, each of which are incorporated herein byreference in their entireties. An exemplary spray-drying methodincludes: combining nitrogen, as a drying gas, with a loading bufferaccording to the present disclosure, then introducing the mixture intoGEA Mobile Minor spray dryer from GEA Processing Engineering, Inc.(Columbia Md., USA), which has a Two-Fluid Nozzle configuration, spraydrying the mixture at an inlet temperature in the range of 150° C. to190° C., an outlet temperature in the range of 65° C. to 100° C., anatomic rate in the range of 0.5 to 2.0 bars, an atomic rate in the rangeof 5 to 13 kg/hr, a nitrogen use in the range of 60 to 100 kg/hr, and arun time of 10 to 35 minutes. The final step in spray drying ispreferentially collecting the dried mixture. The dried composition insome embodiments is stable for at least six months at temperatures thatrange from −20° C. or lower to 90° C. or higher.

TABLE A Exemplary Lyophilization Protocol Temp. Pressure Step Set TypeDuration Set Freezing Step F1 −50° C. Ramp Var N/A F2 −50° C. Hold    3Hrs N/A Vacuum Pulldown F3 −50° Hold Var N/A Primary Dry P1 −40° Hold 1.5 Hrs 0 mT P2 −35° Ramp    2 Hrs 0 mT P3 −25° Ramp    2 Hrs 0 mT P4−17° C. Ramp    2 Hrs 0 mT P5    0° C. Ramp  1.5 Hrs 0 mT P6   27° C.Ramp  1.5 Hrs 0 mT P7   27° C. Hold   16 Hrs 0 mT Secondary Dry S1   27°C. Hold  >8 Hrs 0 mT

In some embodiments, the step of drying the platelets that are obtainedas disclosed herein, such as the step of freeze-drying the plateletsthat are obtained as disclosed herein, includes incubating the plateletswith a lyophilizing agent to generate thrombosomes. In some embodiments,the lyophilizing agent is polysucrose. In some embodiments, thelyophilizing agent is a non-reducing disaccharide. Accordingly, in someembodiments, the methods for preparing thrombosomes from plateletsfurther include incubating the platelets with a lyophilizing agent. Insome embodiments, the lyophilizing agent is a saccharide. In someembodiments, the saccharide is a disaccharide, such as a non-reducingdisaccharide.

In some embodiments, the platelets are incubated with a lyophilizingagent for a sufficient amount of time and at a suitable temperature toload the platelets with the lyophilizing agent. Non-limiting examples ofsuitable lyophilizing agents are saccharides, such as monosaccharidesand disaccharides, including sucrose, maltose, trehalose, glucose (e.g.,dextrose), mannose, and xylose. In some embodiments, non-limitingexamples of lyophilizing agent include serum albumin, dextran, polyvinylpyrrolidone (PVP), starch, and hydroxyethyl starch (HES). In someembodiments, exemplary lyophilizing agents can include a high molecularweight polymer, into the loading composition. By “high molecular weight”it is meant a polymer having an average molecular weight of about orabove 70 kDa. Non-limiting examples are polymers of sucrose andepichlorohydrin. In some embodiments, the lyophilizing agent ispolysucrose. Although any amount of high molecular weight polymer can beused as a lyophilizing agent, it is preferred that an amount be usedthat achieves a final concentration of about 3% to 10% (w/v), such as 3%to 7%, for example 6%.

In some embodiments, the process for preparing a composition includesadding an organic solvent, such as ethanol, to the loading solution. Insuch a loading solution, the solvent can range from 0.1% to 5.0% (v/v).

Within the process provided herein for making the compositions providedherein, addition of the lyophilizing agent can be the last step prior todrying. However, in some embodiments, the lyophilizing agent is added atthe same time or before the anti-fibrinolytic, the cryoprotectant, orother components of the loading composition. In some embodiments, thelyophilizing agent is added to the loading solution, thoroughly mixed toform a drying solution, dispensed into a drying vessel (e.g., a glass orplastic serum vial, a lyophilization bag), and subjected to conditionsthat allow for drying of the solution to form a dried composition.

An exemplary saccharide for use in the compositions disclosed herein istrehalose. Regardless of the identity of the saccharide, it can bepresent in the composition in any suitable amount. For example, it canbe present in an amount of 1 mM to 1 M. In embodiments, it is present inan amount of from 10 mM 10 to 500 mM. In some embodiments, it is presentin an amount of from 20 mM to 200 mM. In some embodiments, it is presentin an amount from 40 mM to 100 mM. In various embodiments, thesaccharide is present in different specific concentrations within theranges recited above, and one of skill in the art can immediatelyunderstand the various concentrations without the need to specificallyrecite each herein. Where more than one saccharide is present in thecomposition, each saccharide can be present in an amount according tothe ranges and particular concentrations recited above.

The step of incubating the platelets to load them with a cryoprotectantor as a lyophilizing agent includes incubating the platelets for a timesuitable for loading, as long as the time, taken in conjunction with thetemperature, is sufficient for the cryoprotectant or lyophilizing agentto come into contact with the platelets and, preferably, beincorporated, at least to some extent, into the platelets. Inembodiments, incubation is carried out for about 1 minute to about 180minutes or longer.

The step of incubating the platelets to load them with a cryoprotectantor lyophilizing agent includes incubating the platelets and thecryoprotectant at a temperature that, when selected in conjunction withthe amount of time allotted for loading, is suitable for loading. Ingeneral, the composition is incubated at a temperature above freezingfor at least a sufficient time for the cryoprotectant or lyophilizingagent to come into contact with the platelets. In embodiments,incubation is conducted at 37° C. In certain embodiments, incubation isperformed at 20° C. to 42° C. For example, in embodiments, incubation isperformed at 35° C. to 40° C. (e.g., 37° C.) for 110 to 130 (e.g., 120)minutes.

In various embodiments, the bag is a gas-permeable bag configured toallow gases to pass through at least a portion or all portions of thebag during the processing. The gas-permeable bag can allow for theexchange of gas within the interior of the bag with atmospheric gaspresent in the surrounding environment. The gas-permeable bag can bepermeable to gases, such as oxygen, nitrogen, water, air, hydrogen, andcarbon dioxide, allowing gas exchange to occur in the compositionsprovided herein. In some embodiments, the gas-permeable bag allows forthe removal of some of the carbon dioxide present within an interior ofthe bag by allowing the carbon dioxide to permeate through its wall. Insome embodiments, the release of carbon dioxide from the bag can beadvantageous to maintaining a desired pH level of the compositioncontained within the bag.

In some embodiments, the container of the process herein is agas-permeable container that is closed or sealed. In some embodiments,the container is a container that is closed or sealed and a portion ofwhich is gas-permeable. In some embodiments, the surface area of agas-permeable portion of a closed or sealed container (e.g., bag)relative to the volume of the product being contained in the container(hereinafter referred to as the “SA/V ratio”) can be adjusted to improvepH maintenance of the compositions provided herein. For example, in someembodiments, the SA/V ratio of the container can be at least about 2.0cm²/mL (e.g., at least about 2.1 cm²/mL, at least about 2.2 cm²/mL, atleast about 2.3 cm²/mL, at least about 2.4 cm²/mL, at least about 2.5cm²/mL, at least about 2.6 cm²/mL, at least about 2.7 cm²/mL, at leastabout 2.8 cm²/mL, at least about 2.9 cm²/mL, at least about 3.0 cm²/mL,at least about 3.1 cm²/mL, at least about 3.2 cm²/mL, at least about 3.3cm²/mL, at least about 3.4 cm²/mL, at least about 3.5 cm²/mL, at leastabout 3.6 cm²/mL, at least about 3.7 cm²/mL, at least about 3.8 cm²/mL,at least about 3.9 cm²/mL, at least about 4.0 cm²/mL, at least about 4.1cm²/mL, at least about 4.2 cm²/mL, at least about 4.3 cm²/mL, at leastabout 4.4 cm²/mL, at least about 4.5 cm²/mL, at least about 4.6 cm²/mL,at least about 4.7 cm²/mL, at least about 4.8 cm²/mL, at least about 4.9cm²/mL, or at least about 5.0 cm²/mL. In some embodiments, the SA/Vratio of the container can be at most about 10.0 cm²/mL (e.g., at mostabout 9.9 cm²/mL, at most about 9.8 cm²/mL, at most about 9.7 cm²/mL, atmost about 9.6 cm²/mL, at most about 9.5 cm²/mL, at most about 9.4cm²/mL, at most about 9.3 cm²/mL, at most about 9.2 cm²/mL, at mostabout 9.1 cm²/mL, at most about 9.0 cm²/mL, at most about 8.9 cm²/mL, atmost about 8.8 cm²/mL, at most about 8.7 cm²/mL, at most about 8.6,cm²/mL at most about 8.5 cm²/mL, at most about 8.4 cm²/mL, at most about8.3 cm²/mL, at most about 8.2 cm²/mL, at most about 8.1 cm²/mL, at mostabout 8.0 cm²/mL, at most about 7.9 cm²/mL, at most about 7.8 cm²/mL, atmost about 7.7 cm²/mL, at most about 7.6 cm²/mL, at most about 7.5cm²/mL, at most about 7.4 cm²/mL, at most about 7.3 cm²/mL, at mostabout 7.2 cm²/mL, at most about 7.1 cm²/mL, at most about 6.9 cm²/mL, atmost about 6.8 cm²/mL, at most about 6.7 cm²/mL, at most about 6.6cm²/mL, at most about 6.5 cm²/mL, at most about 6.4 cm²/mL, at mostabout 6.3 cm²/mL, at most about 6.2 cm²/mL, at most about 6.1 cm²/mL, atmost about 6.0 cm²/mL, at most about 5.9 cm²/mL, at most about 5.8cm²/mL, at most about 5.7 cm²/mL, at most about 5.6 cm²/mL, at mostabout 5.5 cm²/mL, at most about 5.4 cm²/mL, at most about 5.3 cm²/mL, atmost about 5.2 cm²/mL, at most about 5.1 cm²/mL, at most about 5.0cm²/mL, at most about 4.9 cm²/mL, at most about 4.8 cm²/mL, at mostabout 4.7 cm²/mL, at most about 4.6 cm²/mL, at most about 4.5 cm²/mL, atmost about 4.4 cm²/mL, at most about 4.3 cm²/mL, at most about 4.2cm²/mL, at most about 4.1 cm²/mL, or at most about 4.0 cm²/mL. In someembodiments, the SA/V ratio of the container can range from about 2.0 toabout 10.0 cm²/mL (e.g., from about 2.1 cm²/mL to about 9.9 cm²/mL, fromabout 2.2 cm²/mL to about 9.8 cm²/mL, from about 2.3 cm²/mL to about 9.7cm²/mL, from about 2.4 cm²/mL to about 9.6 cm²/mL, from about 2.5 cm²/mLto about 9.5 cm²/mL, from about 2.6 cm²/mL to about 9.4 cm²/mL, fromabout 2.7 cm²/mL to about 9.3 cm²/mL, from about 2.8 cm²/mL to about 9.2cm²/mL, from about 2.9 cm²/mL to about 9.1 cm²/mL, from about 3.0 cm²/mLto about 9.0 cm²/mL, from about 3.1 cm²/mL to about 8.9 cm²/mL, fromabout 3.2 cm²/mL to about 8.8 cm²/mL, from about 3.3 cm²/mL to about 8.7cm²/mL, from about 3.4 cm²/mL to about 8.6 cm²/mL, from about 3.5 cm²/mLto about 8.5 cm²/mL, from about 3.6 cm²/mL to about 8.4 cm²/mL, fromabout 3.7 cm²/mL to about 8.3 cm²/mL, from about 3.8 cm²/mL to about 8.2cm²/mL, from about 3.9 cm²/mL to about 8.1 cm²/mL, from about 4.0 cm²/mLto about 8.0 cm²/mL, from about 4.1 cm²/mL to about 7.9 cm²/mL, fromabout 4.2 cm²/mL to about 7.8 cm²/mL, from about 4.3 cm²/mL to about 7.7cm²/mL, from about 4.4 cm²/mL to about 7.6 cm²/mL, from about 4.5 cm²/mLto about 7.5 cm²/mL, from about 4.6 cm²/mL to about 7.4 cm²/mL, fromabout 4.7 cm²/mL to about 7.3 cm²/mL, from about 4.8 cm²/mL to about 7.2cm²/mL, from about 4.9 cm²/mL to about 7.1 cm²/mL, from about 5.0 cm²/mLto about 6.9 cm²/mL, from about 5.1 cm²/mL to about 6.8 cm²/mL, fromabout 5.2 cm²/mL to about 6.7 cm²/mL, from about 5.3 cm²/mL to about 6.6cm²/mL, from about 5.4 cm²/mL to about 6.5 cm²/mL, from about 5.5 cm²/mLto about 6.4 cm²/mL, from about 5.6 cm²/mL to about 6.3 cm²/mL, fromabout 5.7 cm²/mL to about 6.2 cm²/mL, or from about 5.8 cm²/mL to about6.1 cm²/mL.

Gas-permeable closed containers (e.g., bags) or portions thereof can bemade of one or more various gas-permeable materials. In someembodiments, the gas-permeable bag can be made of one or more polymersincluding fluoropolymers (such as polytetrafluoroethylene (PTFE) andperfluoroalkoxy (PFA) polymers), polyolefins (such as low-densitypolyethylene (LDPE), high-density polyethylene (HDPE)), fluorinatedethylene propylene (FEP), polystyrene, polyvinylchloride (PVC),silicone, and any combinations thereof.

In some embodiments, the lyophilizing agent as disclosed herein may be ahigh molecular weight polymer. By “high molecular weight” it is meant apolymer having an average molecular weight of about or above 70 kDa andup to 1,000,000 kDa Non-limiting examples are polymers of sucrose andepichlorohydrin (polysucrose). Although any amount of high molecularweight polymer can be used, it is preferred that an amount be used thatachieves a final concentration of about 3% to 10% (w/v), such as 3% to7%, for example 6%. Other non-limiting examples of lyoprotectants areserum albumin, dextran, polyvinyl pyrrolidone (PVP), starch, andhydroxyethyl starch (HES). In some embodiments, a lyoprotectant is alsoa cryoprotectant. For example, albumin, polysucrose, and sucrose canalso be used as a cryoprotectant.

In some embodiments, lyophilized platelets can be fixed (e.g.,lyophilized fixed plates) in fixing agent. In some embodiments,lyophilized platelets can be fixed in formalin (e.g., lyophilizedformalin-fixed platelets).

In some embodiments, the lyophilized platelets (e.g., thrombosomes) canbe at a concentration from about 1,000 k/μl to about 500,000 k/μl. Insome embodiments, the lyophilized platelets (e.g., thrombosomes) can beat a concentration from about 5,000 k/μl to about 450,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 10,000 k/μl to about 400,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 30,000 k/μl to about 300,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 40,000 k/μl to about 250,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 50,000 k/μl to about 225,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 60,000 k/μl to about 200,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 70,000 k/μl to about 175,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 80,000 k/μl to about 150,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 90,000 k/μl to about 125,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 100,000 k/μl to about 120,000 k/μl. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 105,000 k/μl to about 115,000 k/μl. In someembodiments, the therapeutically effective amount of lyophilizedplatelets (e.g., thrombosomes) can be at any of the concentrationsdescribed herein).

In some embodiments, the lyophilized platelets (e.g., thrombosomes) canbe at a concentration from about 1,000 to about 500,000 thrombosomes. Insome embodiments, the lyophilized platelets (e.g., thrombosomes) can beat a concentration from about 5,000 to about 450,000 thrombosomes. Insome embodiments, the lyophilized platelets (e.g., thrombosomes) can beat a concentration from about 10,000 to about 400,000 thrombosomes. Insome embodiments, the lyophilized platelets (e.g., thrombosomes) can beat a concentration from about 30,000 to about 300,000 thrombosomes. Insome embodiments, the lyophilized platelets (e.g., thrombosomes) can beat a concentration from about 40,000 to about 250,000 thrombosomes. Insome embodiments, the lyophilized platelets (e.g., thrombosomes) can beat a concentration from about 50,000 thrombosomes to about 225,000thrombosomes. In some embodiments, the lyophilized platelets (e.g.,thrombosomes) can be at a concentration from about 60,000 to about200,000 thrombosomes. In some embodiments, the lyophilized platelets(e.g., thrombosomes) can be at a concentration from about 70,000thrombosomes to about 175,000 thrombosomes. In some embodiments, thelyophilized platelets (e.g., thrombosomes) can be at a concentrationfrom about 80,000 to about 150,000 thrombosomes. In some embodiments,the lyophilized platelets (e.g., thrombosomes) can be at a concentrationfrom about 90,000 to about 125,000 thrombosomes. In some embodiments,the lyophilized platelets (e.g., thrombosomes) can be at a concentrationfrom about 100,000 thrombosomes to about 120,000 thrombosomes. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 105,000 to about 115,000 thrombosomes. In someembodiments, the therapeutically effective amount of lyophilizedplatelets (e.g., thrombosomes) can be at any of the concentrationsdescribed herein).

In some embodiments, the lyophilized platelets (e.g., thrombosomes) canbe at a concentration from about 1×10² particles/kg to from about 1×10¹³particles/kg. In some embodiments, the lyophilized platelets (e.g.,thrombosomes) can be at a concentration from about 1×10³ particles/kg tofrom about 1×10¹² particles/kg. In some embodiments, the lyophilizedplatelets (e.g., thrombosomes) can be at a concentration from about1×10⁴ particles/kg to from about 1×10¹¹ particles/kg. In someembodiments, the lyophilized platelets (e.g., thrombosomes) can be at aconcentration from about 1×10⁵ particles/kg to from about 1×10¹⁰particles/kg. In some embodiments, the lyophilized platelets (e.g.,thrombosomes) can be at a concentration from about 1×10⁶ particles/kg tofrom about 1×10⁹ particles/kg. In some embodiments, the lyophilizedplatelets (e.g., thrombosomes) can be at a concentration from about1×10⁷ particles/kg to from about 1×10⁸ particles/kg. In someembodiments, a therapeutically effective amount of the lyophilizedplatelets (e.g., thrombosomes) can be at any of the concentrationsdescribed herein.

In some embodiments of the methods herein, any of the compositionsdescribed herein are administered topically. In some embodiments,topical administration can include administration via a solution, cream,gel, suspension, putty, particulates, or powder. In some embodiments,topical administration can include administration via a bandage (e.g. anadhesive bandage or a compression bandage) or medical closure (e.g.,sutures, staples)); for example the anti-fibrinolytic loaded plateletderivatives (e.g., lyopreserved platelets (e.g., thrombosomes)) can beembedded therein or coated thereupon), as described in PCT PublicationNo. WO2017/040238 (e.g., paragraphs [013]-[069]), corresponding to U.S.patent application Ser. No. 15/776,255, the entirety of which is hereinincorporated by reference.

In some embodiments of the methods herein, the compositions describedherein are administered parenterally.

In some embodiments of the methods herein, the compositions describedherein are administered intravenously.

In some embodiments of the methods herein, the compositions describedherein are administered intramuscularly.

In some embodiments of the methods herein, the compositions describedherein are administered intrathecally.

In some embodiments of the methods herein, the compositions describedherein are administered subcutaneously.

In some embodiments of the methods herein, the compositions describedherein are administered intraperitoneally. In some embodiments, theanti-fibrinolytic loaded platelets have a storage stability that is atleast about equal to that of the platelets prior to the loading of theanti-fibrinolytic.

The loading buffer may be any buffer that is non-toxic to the plateletsand provides adequate buffering capacity to the solution at thetemperatures at which the solution will be exposed during the processprovided herein. Thus, the buffer may include any of the knownbiologically compatible buffers available commercially, such asphosphate buffers, such as phosphate buffered saline (PBS),bicarbonate/carbonic acid, such as sodium-bicarbonate buffer,N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), andtris-based buffers, such as tris-buffered saline (TB S). Likewise, itmay include one or more of the following buffers:propane-1,2,3-tricarboxylic (tricarballylic); benzenepentacarboxylic;maleic; 2,2-dimethyl succinic; 3,3-dimethylglutaric;bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS);benzenehexacarboxylic (mellitic); N-(2-acetamido)imino-diacetic acid(ADA); butane-1,2,3,4-tetracarboxylic; pyrophosphoric;1,1-cyclopentanediacetic (3,3 tetramethylene-glutaric acid);piperazine-1,4-bis-(2-ethanesulfonic acid) (PIPES);N-(2-acetamido)-2-amnoethanesulfonic acid (ACES);1,1-cyclohexanediacetic; 3,6-endomethylene-1,2,3,6-tetrahydrophthalicacid (EMTA; ENDCA); imidazole; 2-(aminoethyl)trimethylammonium chloride(CHOLAMINE); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);2-methylpropane-1,2,3-triscarboxylic (beta-methyltricarballylic);2-(N-morpholino)propane-sulfonic acid (MOPS); phosphoric; andN-tris(hydroxymethyl)methyl-2-amminoethane sulfonic acid (TES).

A plate reader (e.g., Tecan Microplate reader (e.g., Infinite® 200 PRO))can be used to quantify loading efficiency of the anti-fibrinolytic inthe anti-fibrinolytic loaded platelets. Platelets can be evaluated forfunctionality by adenosine diphosphate (ADP), collagen, arachidonicacid, phorbol myristate acetate (PMA), thrombin receptor activatingpeptide (TRAP), and/or any other platelet agonist known in the art forstimulation post-loading. A hemostasis analyzer (e.g., TEG® 5000Thromboelastogram® Hemostasis Analyzer system) can be used to testanti-fibrinolytic function of EACA loaded platelets.

In some embodiments, the anti-fibrinolytic platelets are lyophilized. Insome embodiments, the anti-fibrinolytic loaded platelets arecryopreserved. In some embodiments, the unloaded platelets arelyophilized. In some embodiments, the unloaded platelets arecryopreserved.

In some embodiments, the anti-fibrinolytic loaded platelets retain theloaded anti-fibrinolytic compound upon rehydration and release theanti-fibrinolytic compound upon stimulation by endogenous plateletactivators, such as endogenous platelet activators described herein.

In some embodiments, the dried platelets (such as freeze-driedplatelets) retain the loaded anti-fibrinolytic upon rehydration andrelease the anti-fibrinolytic (e.g., EACA) upon stimulation byendogenous platelet activators. In some embodiments, at least about 10%,such as at least about 20%, such as at least about 30% of theanti-fibrinolytic is retained. In some embodiments, from about 10% toabout 20%, such as from about 20% to about 30% of the anti-fibrinolyticis retained.

In some embodiments, anti-fibrinolytic loaded platelets,anti-fibrinolytic loaded platelet derivatives, or anti-fibrinolyticloaded thrombosomes can shield the anti-fibrinolytic from exposure incirculation, thereby reducing or eliminating systemic toxicity (e.g.cardiotoxicity) associated with the anti-fibrinolytic. In someembodiments, anti-fibrinolytic loaded platelets, anti-fibrinolyticloaded platelet derivatives, and/or anti-fibrinolytic loadedthrombosomes can also protect the anti-fibrinolytic from metabolicdegradation or inactivation. In some embodiments, anti-fibrinolyticdelivery with anti-fibrinolytic loaded platelets, anti-fibrinolyticloaded platelet derivatives, and/or anti-fibrinolytic loadedthrombosomes can therefore be advantageous in treatment of diseases suchas von Willebrand disease or traumatic bleeding events (e.g.,hemorrhage), since anti-fibrinolytic loaded platelets, anti-fibrinolyticloaded platelet derivatives, and/or anti-fibrinolytic loadedthrombosomes can mitigate systemic side effects. In some embodiments,anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded plateletderivatives, and/or anti-fibrinolytic loaded thrombosomes can be used inany therapeutic setting in which expedited healing process is requiredor advantageous.

In some embodiments, provided herein is a method of treating a diseaseas disclosed herein in a subject in need thereof, comprisingadministering anti-fibrinolytic loaded platelets, anti-fibrinolyticloaded platelet derivatives, or anti-fibrinolytic loaded thrombosomes asdisclosed herein. In some embodiments, provided herein is a method oftreating a disease as disclosed herein in a subject in need thereof,comprising administering cold stored, room temperature stored,cryopreserved thawed, rehydrated, and/or lyophilized platelets, plateletderivatives, or thrombosomes as disclosed herein. In some embodiments,the disease is von Willebrand disease (e.g., any of the von Willebranddiseases disclosed herein).

In some embodiments, unloaded platelets, unloaded platelet derivatives,and/or unloaded thrombosomes can be advantageous in the treatment of adisease. In some embodiments, unloaded platelets, unloaded plateletderivatives, and/or unloaded thrombosomes can be advantageous in thetreatment of disease such as von Willebrand disease.

In some embodiments, provided herein is a method of treating a diseaseas disclosed herein in a subject in need thereof, (e.g., von Willebranddisease), comprising administering to a subject in need thereof,unloaded platelets, unloaded platelet derivatives, or unloadedthrombosomes as disclosed herein. In some embodiments, provided hereinis a method of treating a disease as disclosed herein in a subject inneed thereof, comprising administering unloaded cold stored, roomtemperature stored, cryopreserved thawed, rehydrated, and/or lyophilizedplatelets, unloaded platelet derivatives, or unloaded thrombosomes asdisclosed herein.

While the embodiments of the methods and compositions described hereinare amenable to various modifications and alternative forms, specificembodiments have been shown by way of example in the drawings and aredescribed in detail below. The intention, however, is not to limit themethods and compositions to the particular embodiments described. On thecontrary, the methods and compositions are intended to cover allmodifications, equivalents, and alternatives falling within the scope ofthe methods and compositions as defined by the appended claims.

EXAMPLES Example 1 Thrombosomes in Ristocetin Cofactor Assay

Thrombosomes were tested for their ability to bind von Willebrand factor(vWF) when incubated with ristocetin. The thrombosomes were compared toformalin-fixed platelets as a positive control (FIG. 1). Formalin-fixedplatelets and thrombosomes were incubated with ristocetin and a smallvolume of pooled normal plasma. Agglutination was induced by addingplasma to platelets or thrombosomes and ristocetin. The data show thatthrombosomes in the presence of ristocetin do not aggregate, unlikeformalin-fixed platelets (FIG. 1). 6 different lots (A-F) ofthrombosomes were tested and compared to the fixed platelets. The slopeof the aggregation curve is used to assess the ristocetin co-factorassay, that is, it demonstrates the rate of vWF binding to GPIb. Usingstandardized platelet poor plasma, the assessment only depends on GPIbbinding. The maximum response of thrombosomes was a slope of 15.92 verse61.98 with fixed platelets. The data show that thrombosomes differ fromfixed platelets in their ability to interact with vWF. Without wishingto be limited by any theory, the data suggest that vWF fails tosufficiently bind the GPIb receptor present on the thrombosomes, theGPIb receptor is absent and/or has reduced expression on thrombosomes,or is somehow otherwise inhibited from binding vWF.

Additionally, thrombosomes were assessed for their ability to aggregatein plasma as compared to platelet rich plasma. The assay measures thecapability of platelets and thrombosomes to be activated by ristocetinin plasma. Fresh drawn platelet rich plasma or thrombosomes in plasmaare incubated with ristocetin. FIG. 2 shows that platelets in plasmawere activated and aggregated, whereas thrombosomes in plasma did notactivate and aggregate. The average platelet rich plasma aggregation was48.4%, as compared to thrombosomes in plasma at 1.7%. The data representthrombosomes as a percentage of “positive” groupthrombosomes=1.7/48.4=3.5% magnitude vs platelet rich plasma. The datain FIG. 1 and FIG. 2 show that thrombosomes are do not interact with vWFin a significant amount to cause aggregation in either the ristocetinco-factor assay or ristocetin-induced platelet activity assay,respectively. Without wishing to be bound by any theory, the lack ofinteraction between thrombosomes and vWF may be due to loss of GPIb(CD42b) receptor expression.

CD42b is the portion of the CD42 protein receptor that binds vWF.Specific antibody clone AN51 also binds the CD42b domain of the receptorand can block vWF binding and subsequent tethering to collagen byplatelets as reported by Dong et al., Ristocetin-dependent, but notbotrocetin-dependent, binding of von Willebrand factor to the plateletglycoprotein ¹b-IX-V complex correlates with shear-dependentinteractions, Blood, 97, 162-168 (2001). AN51 antibody will inhibit boththe Ristocetin Co-Factor assay (FIG. 1) and Ristocetin plateletactivation assay (FIG. 2) mimicking the activity of thrombosomes. Flowcytometry staining of surface expression of CD42b thrombosomes are shownto express far less of this receptor as compared to normal platelets(FIG. 3). The data demonstrate another example of the lack ofinteraction between vWF and thrombosomes.

Protocol—Ristocetin Co-factor Assay

The Ristocetin Co-factor assay determines whether thrombosomesagglutinate in response to ristocetin+vWF. A positive response suggestsintact, functional GP1bα receptors on the surface of the thrombosomes.

Materials/Reagents:

-   -   Helena Hemostasis Ristocetin Cofactor Assay (RCA) Kit (Cat        #5370; Lot 2-18-5370,        https://www.helena.com/Procedures/Pro064Rev5.pdf)        -   ristocetin (10 mg/mL reconstituted)        -   formalin-fixed platelets    -   0.2 um filtered George King normal human plasma    -   Corning Cell Culture Grade 1×PBS    -   Corning Cell Culture Grade Water    -   Helena Laboratories AggRAM system (1-158-0000)    -   Aggregometer cuvettes    -   Aggregometer stir bars    -   Micropipettes & tips    -   1 unit Thrombosomes    -   Beckman-Coulter AcT diff 2 (1-418-0000)

Protocol:

-   -   1. Start the AggRAM as described in EQU-031 and perform the        daily optical calibration check. There are ristocetin cofactor        assay run settings built into the HemoRAM software. Use spin        speed 600 rpm for all runs.    -   2. Bring RCA kit components to room temperature and rehydrate as        indicated on the vial label.        -   a. Reconstitute ristocetin with 1.5 mL water, swirl gently            and allow to stand 10 minutes.    -   3. Prepare Thrombosomes samples by rehydrating Thrombosomes as        indicated in PRO-022.        -   a. Take 2×AcT counts of rehydrated Thrombosomes.        -   b. Dilute aliquots washed Thrombosomes as needed to            concentrations of:            -   i. ˜375 k/μL unwashed Thrombosomes in PBS.            -   ii. ˜375 k/μL washed Thrombosomes in PBS.    -   4. Prepare the 100% activity “blank” by diluting Thrombosomes        1:2 in PBS (final volume 250 μL) in an aggregometry cuvette with        stir bar.    -   5. Prepare samples by adding 200 μL of Thrombosomes suspension        to aggregometry cuvettes with stir bar.    -   6. Warm sample and blank cuvettes at 37° C. in the holding wells        for 5 minutes prior to inserting in the aggregation wells.    -   7. Insert the “blank” cuvette into the first aggregation well        and press the channel button. Allow to blank and replace with        the first sample cuvette. Repeat for each sample/channel.    -   8. Add 25 μL ristocetin to the sample cuvette and press the        channel button. Repeat for each sample, and allow to equilibrate        1 minute.    -   9. Add 25 μL filtered plasma to the sample cuvette and press the        channel button. Repeat for each sample. Record runs for 5        minutes.    -   10. Export PDF of RCA TopChart output to appropriate file        location.        Note: Formalin-fixed platelet positive controls were established        at start of day to ensure ristocetin, GK plasma elicit        appropriate agglutination response.

Sample Setup:

Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 Channel 7Channel 8 Unwashed Unwashed Unwashed Unwashed Washed Washed WashedWashed PBS Plasma Plasma Plasma PBS Plasma Plasma PlasmaVolume each Thrombosomes dilution needed=[200(4)+125]*1.25=1.2 mLVolume ristocetin needed per batch=8*25*1.25=250 μL

(total 2×1.5 mL bottles rehydrated ristocetin for n=12)

Volume GK Plasma needed per batch=6*25*1.25=200

(total 3×1 mL aliquots plasma filtered for n=12)

Total n=2 vials for each of 6 batches.

Flow Cytometry Assay:

Pooled apheresis platelet product (from 3 units, 24 hours postcollection)

Standard Loading Buffer (pH 6.5-6.8)

30% Polysucrose

St Gobain VueLife 32C FEP bags

Wheaton 5 ml vials

Manufacturing Disposables

Test Conditions:

Sublot A: Standard Thrombosomes®

Sublot B: CPP Optimized Thrombosomes®

-   -   Final formulation contains 1% DMSO, 1% Glycerol, and 10%        Polysucrose

Methods:

-   -   1. Acidify platelets to pH ˜6.6 with citric acid. Centrifuge PRP        at 1250 g for 20 min.    -   2. Re-suspend the platelet pellets in standard loading buffer.        Target a count of 2500×10³ plts/μl (prepare ˜40 ml total).    -   3. Transfer the platelets to a FEP bag. Incubate at 37 C with        agitation for 3 hours    -   4. Add ¼ volume 30% Polysucrose to achieve 6% Polysucrose final        concentration.    -   5. Take an aliquot from each sub-lot for pre-lyophilized        testing. Each sub-lot will be tested by:        -   a) Flow cytometry (surface markers, morphology, & count)    -   6. Fill each sub-lot into pre-labeled 5 ml vials with 1 ml        fills. Add a stopper and transfer the vials to either the        Stellar lyophilized with a pre-chilled shelf (−50 C) or the −80        C freezer shelf. Lyophilize the product using the following        recipe:        -   Freezing:            -   Step 1: Ramp up to −50 C for 0 minutes.            -   Step 2: −50 C for 180 minutes.        -   Final Freezing:            -   Shelf −40 C at 0 minutes; pressure at 0 mT.        -   Primary Drying:            -   1: Ramp shelf to −30 for 120 minutes (5 C/hr)            -   Step 2: Hold shelf at −30 for 2880 minutes            -   Step 3: Ramp shelf to −10 for 240 minutes (5 C/hr)            -   Step 4: Hold shelf at −10 for 2880 minutes            -   Step 3: Ramp shelf to +25 for 420 minutes (5 C/hr)        -   Secondary Drying:            -   Shelf 25° C. for 720 minutes; pressure at 0 mT.            -   Shelf 25° C. at 9999 minutes; pressure at 0 mT. Hold for                a minimum of 1 hour.    -   7. Stopper and cap all the vials following completion of the        lyophilization cycle.    -   8. Bake 80 C for 24 hours.    -   9. Test 1 vial by:        -   a) Flow cytometry (surface markers, morphology, & count)

Flow Protocol:

Counts

Goal—Determine the cell concentration of the pre-lyophilized materialfrom each sub-lot

-   -   1. For each sub-lot prepare the following dilutions in        duplicate.    -   2. For each pre-lyophilized sample add 10 μL of sample to 990 μL        of PBS. Thoroughly mix the sample by pipetting before adding 100        μL of diluted sample to 900 μL of PBS. This will generate a        pre-lyophilized sample with a final dilution factor of 1:1000.    -   3. Transfer 100 μL of each sample to an individual well on a 96        well plate.    -   4. Acquire each sample on the NovoCyte with the following        conditions:        -   a. Parameters: FSC, SSC        -   b. Stop Conditions: 50 μL or 30,000 events        -   c. FSC-H Threshold @1,000        -   d. Absolute count dilution: 1,000    -   5. Determine the concentration of the platelet size population        for each sample.

Size and Surface Marker Testing

This assay tests the size distribution and surface marker positivity ofa sample at a standard concentration for the pre lyophilized materialfrom each sub-lot. Additionally, each sample was single stained forCD42b using a PE conjugated anti-CD42b antibody (AN51).

-   -   1. Based on the flow count obtained in the previous section,        create a 400 μL dilution of each sub-lot in PBS. The final count        for each dilution should be 1,000,000 per μL.    -   2. For each sub-lot create the following 1:10 dilutions:        -   a. Cells without calcium and GPRP: 225 μL HMTA+25 μL cells        -   b. Cells with calcium and GPRP: 24 μL 150 mM CaCl₂+2 μL            GPRP+199 μL HMTA+25 μL cells    -   3. Create HBS with 3 mM CaCl₂ by adding 160 μL of 150 mM CaCl₂        to 7,800 μL HBS.    -   4. Prepare staining mixes according to the tables below. This        will provide enough antibody to stain the hIDSP and both        sub-lots of pre-lyophilized material.

CD62P Iso HN/ITA 14 Anti-CD41 PE 84 mIgG1 PECy5 35 CD62P Test HN/ITA 14Anti-CD41 PE 84 Anti-CD62P 35 PECy5 AV HMTA 64 Anti-CD41 PE 156 AV BV71126

-   -   5. Generate the following samples in duplicate for each        pre-lyophilized sample:        -   a. Unstained: 19 μL HMTA+5 μL cells        -   b. CD62P Iso: 19 μL CD62P Iso mix+5 μL cells        -   c. CD62P Test: 19 μL CD62P Test Mix+5 μL cells        -   d. AV Neg: 19 μL AV Mix+5 μL cells        -   e. AV Pos: 19 μL AV Mix+5 μL cells with calcium and GPRP        -   f. CD42b: 17 μL HMTA+2 μL anti-CD42b+5 μL cells    -   6. Incubate all samples away from open light at room temperature        for 20 minutes.    -   7. After incubation, add 400 μL HBS to each sample. Use HBS        containing calcium to dilute AV test samples.    -   8. Transfer 100 μL of each sample to an individual well in a 96        well plate.    -   9. Acquire each sample on the NovoCyte with the following        conditions:        -   a. Parameters: FSC, SSC, B572, B660, V725        -   b. Stop Conditions: 25 μL or 20,000 events        -   c. FSC-H Threshold @1,000        -   d. Flow Rate: Medium

Example 2—Thrombosome Clotting In Vitro

FIG. 4 shows that normal platelets and thrombosomes clot similarly undershear force upon exposure to collagen and tissue factor. Despite thelimited interaction between vWF and thrombosomes (FIGS. 1-3), thelimited interaction does not inhibit the ability of thrombosomes to formclots as measured by the T-TAS system. In the T-TAS system, thrombosomesand fresh platelets in normal plasma formed similar clots under shearforces stimulated by tissue factor and collagen coated channel (FIG. 4).The data support that thrombosomes are capable of clot formation undershear stress and coagulation activation. The lack of aggregation bythrombosomes in the presence of ristocetin and yet normal clot formationdemonstrate that thrombosomes can form clots in the absence of vWF.

Next, thrombosomes were assayed for their ability to form clots in vWFdeficient plasma. Thrombosomes in normal plasma cause the occlusion ofcollagen and tissue factor channel on the AR chip as measured by theT-TAS system. Thrombosomes from the same lot were assayed vWF deficientplasma and occluded similarly to normal plasma (FIG. 5), demonstratingthrombosomes ability to form clots without the involvement of vWF.

The T-TAS® instrument was prepared for use according to themanufacturer's instructions. AR Chips (Diapharma Cat. #TC0101,http://diapharma.com/wp-content/uploads/2016/03/DiaPharmaProductList_ML-00-00002REV7.pdf)and AR Chip Calcium Corn Trypsin Inhibitor (CaCTI; Diapharma Cat.#TR0101,http://diapharma.com/wp-content/uploads/2016/03/DiaPharmaProductList_ML-00-00002REV7.pdf)were warmed to room temperature. 300 μL of rehydrated thrombosomes weretransferred to a 1.7 mL microcentrifuge tube and centrifuged at 3900 gfor 10 minutes to pellet. The thrombosome pellet was resuspended innormal human plasma or autologous plasma with or without autologousplatelets to a concentration of approximately 100,000-450,000thrombosomes/μL, as determined by AcT counts (Beckman Coulter AcT Diff 2Cell Counter). 20 μL of CaCTI with 480 μL of thrombosomes sample inplasma sample were mixed with gentle pipetting. The sample was loadedand run on the T-TAS® according to the manufacturer's instructions.

Embodiments

Embodiment 1 is a method of treating von Willebrand disease in asubject, the method comprising: administering a therapeuticallyeffective amount of unloaded thrombosomes to the subject in needthereof.

Embodiment 2 is a method of treating von Willebrand disease in asubject, the method comprising: administering a therapeuticallyeffective amount of thrombosomes to the subject, wherein the method doesnot comprise administering an anti-fibrinolytic.

Embodiment 3 is the method of embodiment 1 or 2, wherein the vonWillebrand disease is von Willebrand disease type 1, von Willebranddisease type 2, or von Willebrand disease type 3.

Embodiment 4 is the method of embodiment 1 or 2, wherein the vonWillebrand disease is acquired von Willebrand disease.

Embodiment 5 is the method of any one of embodiments 1 to 4, wherein theconcentration of the therapeutically effective amount of unloadedthrombosomes is from about 1×10² particles/kg to about 1×10¹³particles/kg.

Embodiment 6 is the method of any one of embodiments 1-5, wherein theconcentration of the therapeutically effective amount of unloadedthrombosomes is from about 1×10⁴ to about 1×10¹¹ particles/kg.

Embodiment 7 is the method of any one of embodiments 1-6, wherein theconcentration of the therapeutically effective amount of unloadedthrombosomes is from about 1×10⁶ to about 1×10⁹ particles/kg.

Embodiment 8 is the method of any one of embodiments 1-4, wherein theconcentration of the therapeutically effective amount of unloadedthrombosomes is at least 8.5×10⁸ particles/kg.

Embodiment 9 is the method of any one of embodiments 1-4 and 8, whereinthe concentration of the therapeutically effective amount of unloadedthrombosomes is at least 8.49×10⁹ particles/kg.

Embodiment 10 is the method of any one of embodiments 1-9, wherein thesurface expression of CD42b on the therapeutically effective amount ofunloaded thrombosomes is about 50% less than the surface expression ofCD42b on platelets.

Embodiment 11 is the method of any one of embodiments 1-10, wherein thesurface expression of CD42b on the therapeutically effective amount ofunloaded thrombosomes is about 40% less than the surface expression ofCD42b on platelets.

Embodiment 12 is the method of any one of embodiments 1-11, wherein thesurface expression of CD42b on the therapeutically effective amount ofunloaded thrombosomes is about 25% less than the surface expression ofCD42b on platelets.

Embodiment 13 is the method of any one of embodiments 1-12, wherein thetherapeutically effective amount of unloaded thrombosomes forms clots invon Willebrand factor deficient plasma.

Embodiment 14 is the method of any one of embodiments 1-13, wherein thetherapeutically effective amount of unloaded thrombosomes areadministered topically.

Embodiment 15 is the method of any one of embodiments 1-13, wherein thetherapeutically effective amount of unloaded thrombosomes areadministered intravenously.

Embodiment 16 is the method of any one of embodiments 1-13, wherein thetherapeutically effective amount of unloaded thrombosomes areadministered intramuscularly.

Embodiment 17 is the method of any one of claims 1-13, wherein thetherapeutically effective amount of unloaded thrombosomes areadministered subcutaneously.

1. A method of treating von Willebrand disease in a subject, the methodcomprising: administering a therapeutically effective amount offreeze-dried platelets to the subject in need thereof.
 2. A method oftreating von Willebrand disease in a subject, the method comprising:administering a therapeutically effective amount of freeze-driedplatelets to the subject, wherein the method does not compriseadministering an anti-fibrinolytic.
 3. The method of claim 1, whereinthe von Willebrand disease is von Willebrand disease type 1, vonWillebrand disease type 2, or von Willebrand disease type
 3. 4. Themethod of claim 1, wherein the von Willebrand disease is acquired vonWillebrand disease.
 5. The method of claim 1, wherein the concentrationof the therapeutically effective amount of freeze-dried platelets isfrom about 1×10² particles/kg to about 1×10¹³ particles/kg.
 6. Themethod of claim 1, wherein the concentration of the therapeuticallyeffective amount of freeze-dried platelets is from about 1×10⁴ to about1×10¹¹ particles/kg.
 7. The method of claim 1, wherein the concentrationof the therapeutically effective amount of freeze-dried platelets isfrom about 1×10⁶ to about 1×10⁹ particles/kg.
 8. The method of claim 1,wherein the concentration of the therapeutically effective amount offreeze-dried platelets is at least 8.5×10⁸ particles/kg.
 9. The methodof claim 1, wherein the concentration of the therapeutically effectiveamount of freeze-dried platelets is at least 8.49×10⁹ particles/kg. 10.The method of claim 1, wherein the surface expression of CD42b on thetherapeutically effective amount of freeze-dried platelets is about 50%less than the surface expression of CD42b on platelets.
 11. The methodof claim 1, wherein the surface expression of CD42b on thetherapeutically effective amount of freeze-dried platelets is about 40%less than the surface expression of CD42b on platelets.
 12. The methodof claim 1, wherein the surface expression of CD42b on thetherapeutically effective amount of freeze-dried platelets is about 25%less than the surface expression of CD42b on platelets.
 13. The methodof claim 1, wherein the therapeutically effective amount of freeze-driedplatelets forms clots in von Willebrand factor deficient plasma.
 14. Themethod of claim 1, wherein the therapeutically effective amount offreeze-dried platelets are administered topically.
 15. The method ofclaim 1, wherein the therapeutically effective amount of freeze-driedplatelets are administered intravenously.
 16. The method of claim 1,wherein the therapeutically effective amount of freeze-dried plateletsare administered intramuscularly.
 17. The method of claim 1, wherein thetherapeutically effective amount of freeze-dried platelets areadministered subcutaneously.
 18. A method of treating a coagulopathy ina subject, the method comprising administering to the subject in needthereof a therapeutically effective amount of a composition comprisingplatelets or platelet derivatives and an incubating agent comprising oneor more salts, a buffer, optionally a cryoprotectant, and optionally anorganic solvent, wherein the composition is administered to the subjecthaving von Willebrand disease.
 19. (canceled)
 20. The method of claim18, wherein the von Willebrand disease is von Willebrand disease type 1,von Willebrand disease type 2, von Willebrand disease type 3, oracquired von Willebrand disease.